The use of in vitro cell culture systems is critical to the field of cell biology and to the understanding of the mechanisms of action underlying human diseases. A wide variety of mammalian cell types have been isolated from mammalian organs and tissues, either from normal tissues or from disease-state tissues, such as tumors and metastases. Different cell types often have individual nutritional requirements and therefore, many distinct media have been developed specifically to meet the nutritional requirement of the different cell types. Many of these media contain sera as an additive, such as FBS (fetal bovine serum), HS (horse serum) and CS (calf serum). The use of serum can be cost prohibitive when culturing cells on a large-scale. Furthermore, the use of serum can be problematic, even on the small-scale, as it contains a number of uncharacterized ingredients that can vary from lot to lot. Additionally, serum contains growth-inhibitory and differentiating factors, as well as de-stabilizing the genetic material leading to genetic changes and eventually senescence (Loo et al., Science 236 (4798):200-2 (Apr. 10, 1987)) of the cells.
The use of a serum-free, chemically defined medium solves all of these problems. F-12:DME (1:1 v/v) was originally developed for the propagation of cells in a chemically defined, serum-free system (Mather et al, Experimental Cell Research 120: 191-200,1979; Bottenstein et al, Methods in Enzymology 58: 94-109, 1979). This media can be used with a wide variety of cells in conditions with or without sera. Media NCTC-135 is another media formulated for serum-free cell culture growth (Evans et al, Experimental Cell Research 36: 439-448, 1964).
Tsao et al. formulated a nutrient medium—designated MCDB 152—supplemented with specified growth factors and hormones for the growth of human epidermal cells (Tsao, M. C. et al., J Cellular Physiology. 110:219-229 (1982)). Further refinements of this medium lead to the development of a medium known as MCDB 153 (see Boyce, S. T. and Ham, R. G., J. Invest. Dermatol. 81:33-40 (1983)). The use of these media lead to a more accurate characterization of the necessary growth factors, hormones and Ca.sup.2+ requirements for retention of high cloning efficiency which is necessary to maintain proper genetic programming for continued subculture of pluripotent basal epidermal stem cells. See also Wille, J. J. et al., J Cellular Physiol. 121:31-44 (1984) and U.S. Pat. No. 5,834,312, and the various media discussed therein.
Many media for growing cells in culture are described in the literature or are commercially available. Deriving an optimal medium for specific cell culture needs can lead to improvements in cell growth including increased growth rates, growth to high cell densities, controlling the stage and amount of cell differentiation, increasing protein secretion, increased phenotypic and genetic stability, and elimination of senescence for many cell types.
In optimizing cell culture media for growth, modulating the glucose levels can change the ability of cells to grow in a high cell density condition. Glucose levels are also important to the rate of cell proliferation. As cells metabolize in culture, lactic acid is often produced as a by-product of glycolysis. This leads to a rapid drop in pH, eventually causing cell death. The use of fructose in cell culture media as the major carbon energy source theoretically would lead to the decreased production of lactic acid.
Several researchers have compared the metabolic effects of fructose and glucose on growth and morphological aspects of cells such as human fibroblasts. Others have compared fructose and galactose. For example, Delhotal et al. reported four to five times less lactate production when fructose was used instead of glucose in culturing human skin fibroblast and liver cells (In Vitro 20(9): 699-706, 1984). See, also, Delhotal et al., In Vitro Cell Dev Biol. 23(5): 355-60 (May 1987); Wolfrom et al., Exp Cell Res. 149(2):535-46 (December 1983); Barngrover et al., J Cell Sci 78:173-89 (1985); Low et al., Dev Biol Stand 60:73-9 (1985). See also Mochizuki et al., Cytotechnology 13(3): 161-73 (1993), that compares the use cell culture media containing either fructose or glucose for production of human monoclonal antibodies. Despite these research efforts, glucose is still the major carbon energy source in most commercially available cell culture formulations.
While the majority of cell culture studies use established cell lines, the use of primary cell culture is preferable in some cases. Quite often established cell lines are derived from disease-state tissues such as tumors or cells that have undergone chromosomal changes. In order to study properties of cells in vitro that are recently removed from an in vivo situation, primary culture is often needed. Given the complex interactions between cells in an organ or tissue, deriving a primary culture of one specific cell type isolated from tissues can be problematic. Establishing a primary culture of one specific cell type, however, can be achieved through optimization of the nutrients, attachment factors and growth factors present in the culture medium.
Accordingly, it is an object of this invention to prepare a culture media that supports the growth of primary mammalian cell culture from a variety of tissues. Another object of this invention is to provide a culture media that is serum-free and can be used in both small-scale and large-scale cell culture. A cell culture medium that is versatile, yet defined, would be extremely valuable for the purpose of creating new cell lines and resources from a variety of primary tissues.